A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis

被引：34

作者：

Davis, Richard P. ^{[1
]}

Costa, Magdaline ^{[1
]}

Grandela, Catarina ^{[1
]}

Holland, Andrew M. ^{[1
]}

Hatzistavrou, Tanya ^{[1
]}

Micallef, Suzanne J. ^{[1
]}

Li, Xueling ^{[1
]}

Goulburn, Adam L. ^{[1
]}

Azzola, Lisa ^{[1
]}

Elefanty, Andrew G. ^{[1
]}

Stanley, Edouard G. ^{[1
]}

机构：

[1] Monash Univ, STRIP 1, Monash Immunol & Stem Cell Labs, Clayton, Vic 3800, Australia

来源：

NATURE PROTOCOLS | 2008年 / 3卷 / 10期

基金：

英国医学研究理事会;

关键词：

D O I：

10.1038/nprot.2008.146

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take similar to 1 month.

引用

页码：1550 / 1558

页数：9

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